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Expression analysis by RT-PCR of genes involved in ethylene synthesis and signal transduction in miniature roses
Scientia Horticulturae 216 (2017) 22–28
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Expression analysis by RT-PCR of genes involved in ethylene synthesis and signal transduction in miniature roses Mohammad Al-Salem, Margrethe Serek ∗ Leibniz University of Hannover, Faculty of Natural Sciences, Institute of Horticulture Production Systems, Floriculture, Herrenhaeuser Str. 2, D-30419 Hanover, Germany
a r t i c l e
i n f o
Article history: Received 30 September 2016 Received in revised form 27 December 2016 Accepted 29 December 2016 Keywords: Ethylene receptor Ethylene sensitivity Gene expression Postharvest Rosa hybrida L. Signal transduction
a b s t r a c t Ethylene is one of many factors that affect the quality, appearance and longevity of miniature roses. In this study RT-PCR is used to compare the expression of ethylene biosynthetic genes and ethylene signal transduction genes at different stages of flower development in the two cultivars ‘Vanilla’ and ‘Lavender’, which show low and high ethylene-sensitivity, respectively, and their F1 offspring. The genes for the ethylene receptors RhETR1, RhETR2 and RhETR3, the genes for the receptor-associated signaling proteins RhCTR1 and RhCTR2, the genes for the transcription factors RhEIN3 and RhEIL, and the genes for the ACC synthases RhACS1 and RhACS2 each had an expression pattern that varied between the tested plants and tissues, but could not be correlated with the ethylene sensitivity of the plants. RhETR1, RhETR2, RhETR3 and RhEIN3 were, e.g., expressed more in ‘Vanilla’ than in ‘Lavender’ in most, but not all, of the investigated tissues, but were in general not expressed more in progeny with low sensitivity than in progeny with high sensitivity. No expression was detected for the 4 other genes that were investigated, i.e. genes for RhETR4, RhACS3, RhACS4 and RhACS5. It is concluded that the precise transcriptional activities of the tested genes do not appear to be crucial in determining the ethylene sensitivity of miniature roses. It therefore appears likely that transcription of other genes involved in ethylene signal transduction, posttranscriptional or posttranslational control, or crosstalk with other signal transduction pathways may be important for the degree of ethylene sensitivity of miniature roses. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Miniature rose, Rosa hybrida L., is one of the most economically important ornamental plants for indoor use. Miniature potted roses have become very popular in the last two decades due to their availability in a wide range of colors, sizes, growth habits, and to improved postharvest performance. The annual world production is estimated to be more than 100 million plants, and Denmark is the largest producer with nearly 50% of the world’s production (Pemberton et al., 2003). The number of miniature rose plants sold in 2015 at Flora Holland, the world largest flower auction, reached 47 million, generating revenues of EUR 57 million (FloraHolland, 2016). Ethylene is one of the most important factors affecting the quality, appearance and longevity of many ornamentals (Serek et al., 2006; Ferrante et al., 2015). In miniature roses, ethylene can cause unwanted effects such as premature and accelerated wilting and
∗ Corresponding author. E-mail address: [email protected] (M. Serek). http://dx.doi.org/10.1016/j.scienta.2016.12.029 0304-4238/© 2016 Elsevier B.V. All rights reserved.
abscission of leaves, floral buds, petals and flowers, as well as leaf yellowing or discoloration (Serek 1993; Andersen et al., 2004). The display life of some commercially grown miniature potted rose cultivars was compared by Müller et al. (1998), and it was found that it varied considerably, at least partly due to differences in endogenous ethylene production and sensitivity to exogenous ethylene. In higher plants, the mechanism of ethylene biosynthesis has been well investigated (Yang and Hoffman, 1984; Xu and Zhang 2015). Ethylene signal transduction has also been investigated extensively, resulting in a model encompassing the following wellestablished aspects (Merchante et al., 2013; Cho and Yoo 2015; Gallie 2015; Shakeel et al., 2015). In the absence of ethylene, the ethylene receptors, located in the ER membrane, activate CTR1 (and its homologs) which is associated with the receptors and, when activated, phosphorylates and inhibits EIN2 (a membrane protein also located in the ER membrane). In the presence of ethylene, CTR1 is “switched off”, and EIN2 becomes dephosphorylated, leading to release of its C-terminal domain (Ju et al., 2012). This domain translocates to the nucleus, where it blocks degradation of the transcription factors EIN3/EIL1 and thus activates the transcriptional response to ethylene. Other, less well-characterized proteins,
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also are involved in the signal transduction pathway (Cho and Yoo 2015). There are several ethylene receptors (5 in Arabidopsis) with partly overlapping function (Shakeel et al., 2013; Gallie 2015). From this model, it may be expected that increased amounts of receptor and CTR1 proteins would lead to less ethylene-sensitive plants, whereas increased amounts of EIN2 and/or EIN3/EIL1 proteins would lead to more ethylene-sensitive plants. In attempts to clarify the molecular causes for the differences in flower longevity among miniature rose cultivars, the expression of transcripts for ethylene biosynthetic genes, ethylene receptor genes and other ethylene signal transduction genes have been investigated (Müller et al., 2000a, 2000b, 2001, 2002, 2003; Xue et al., 2008; Ahmadi et al., 2009). For miniature roses, sequences from 4 ethylene receptor genes, namely RhETR1, RhETR2, RhETR3, and RhETR4, have been isolated by Müller et al. (2000a, b), and a fifth gene, RhETR5, was identified by Tan et al. (2006). Mibus and Serek (2004) isolated sequences from 5 ACC synthase genes and Wang et al. (2004) isolated 7 ACC synthase cDNAs. Two genes for ethylene receptor-associated kinases have been identified in miniature roses, namely RhCTR1 and RhCTR2 (Müller et al., 2002). In 2003 Müller et al. identified a gene with high homology to the Arabidopsis gene for the transcription factor EIN3 (also involved in ethylene signal transduction). This gene is in the present work referred to as RhEIL (EIN3-like). In 2006 Tan et al. identified another EIN3-like sequence (here termed RhEIN3) in cut roses. As reported by Müller et al. (1998) ‘Vanilla’ is a cultivar with low sensitivity to ethylene. This cultivar has later been crossed to the highly sensitive cultivar ‘Lavender’ (Buanong et al., 2005; Ahmadi et al., 2009). Some F1 plants (genotypes 50 and 74) were more sensitive to ethylene than ‘Lavender’ while some (genotypes 48 and131) were less sensitive to ethylene than ‘Vanilla’ as judged from the rate of bud or leaf abscission during 15 days of exposure to ethylene. The expression of RhETR1, RhETR3, RhCTR1 and RhCTR2 transcripts in pedicels and petioles were tested in these F1 plants and the parent cultivars after 3 days of ethylene exposure, but no correlation to ethylene sensitivity was found (Ahmadi et al., 2009). Observations on ethylene sensitivity and receptor expression that are in accordance with the current model of ethylene signaling, have been reported by Tieman et al. (2000), who found that transgenic tomatoes with reduced LeETR4 gene expression show high ethylene sensitivity, by Wuriyanghan et al. (2009), who reported that overexpression of the ethylene receptor gene OsETR2 in rice reduces ethylene sensitivity, and by Cancel and Larsen (2002), who found that mutational loss of the ethylene receptor ETR1 in Arabidopsis results in a significant increase in sensitivity to ethylene. In the present study, different tissues from two cultivars ‘Vanilla’ and ‘Lavender’, which show low and high ethylene-sensitivity, respectively, and their F1 offspring were tested by RT-PCR at different developmental stages for transcripts of 8 ethylene transduction genes (RhETR1-4, RhCTR1-2, RhEIN3 and RhEIL) and 5 ACC synthase genes (RhACS1-5) in order to correlate ethylene sensitivity with expression of one or more of these genes and thus explain the differences between postharvest life of the tested cultivars.
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than ‘Vanilla’ and genotype 50 and 74 showed higher ethylenesensitivity than ‘Lavender’. The plants were grown in greenhouses under the following conditions: 22 ◦ C day/20 ◦ C night temperatures and 60–85% relative humidity with 350 mol m−2 s−1 of light from SON-T lamps (Osram, 400 W, Philips, Eindhoven, The Netherlands) with a supplement of natural daylight over a 16 h photoperiod. Total RNA was isolated from selected plant tissues and stages of development. Expression patterns for the genes investigated were determined in carpels and petals from stage 1–3 flowers (Fig. 1), floral buds, and leaves of ‘Vanilla’ and ‘Lavender’, whereas, for the F1 plants, it was investigated in petals of the fully blooming stage (stage 2) flower floral buds and leaves. 2.2. Total RNA extraction and cDNA synthesis A modified hot borate method (Wilkins and Smart, 1996) was used to isolate a high yield of total RNA. Two g of finely ground, frozen plant material of each kind were mixed with 8 mL of the 80–90 ◦ C hot extraction buffer (1% SDS, 1% sodium deoxycholic acid, 5 mM EDTA, 0.2 M Borax (Sodium borate decahydrate)) in 50 mL tubes. PVP (2%), DTT (1%), and Np-40 (1%) had been added to the extraction buffer just before heating. 110 L of Proteinase K (20 mg/mL) was added to each tube. The tubes were incubated at 42 ◦ C for 90 min in a water bath, and were vortexed each 10 min. Then the tubes were removed from water bath and 2 M KCl was added to a final concentration of 160 mM KCl. The tubes were shaken vigorously and incubated 1 h on ice. After that the tubes were centrifuged at 10000g for 20 min at 4 ◦ C. After centrifugation, the aqueous phase was transferred to 20 mL tubes and 8 M LiCl was added to a final concentration of 2 M LiCl. The tubes were placed in a refrigerator (4 ◦ C) overnight. The following day, RNA was precipitated by centrifugation at 14000g at 4 ◦ C for 20 min. The supernatants were discarded and the pellets were washed in 4 mL 2 M LiCl and suspended in 2 mL 10 mM Tris-HCl (pH 7.5) by gentle vortexing. Insoluble materials were removed from the RNA pellet by centrifugation at 10000g for 10 min at 4 ◦ C. After several steps of washing and centrifuging, the pellet was dissolved in 300 L DEPC-treated water. DNase I treatment was performed as described by manufacture (Fermentas GmbH, Germany) to degrade contaminating genomic DNA. First strand complementary DNA (cDNA) was prepared using M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant kit (Promega, USA). One g of total RNA was mixed with 1 g of the Oligo dT (23) primer in a total volume of 7 L. The mixture was heated to 70 ◦ C for 5 min using a T3 thermocycler (Biometra, Germany) and quickly cooled on ice for 5 min. Then the following components were added to the annealed primer in the order: M-MLV RT Reaction buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 , 10 mM DTT) (Promega, USA), 0.5 mM dNTP, 100 units M-MLV RT (H-) (Promega, USA) and 20 units of RNase out (Fermentas GmbH, Germany) in a total volume of 12.5 L. This reaction mixture was incubated for 10 min at 40 ◦ C followed by 150 min at 50 ◦ C and finally by 15 min at 70 ◦ C for deactivation of the reverse transcriptase.
2. Materials and methods
2.3. PCR primer design and optimization of number of PCR cycles
2.1. Plant material
The primer pairs used in this study are summarized in Supplementary Table S1. The primers were designed by use of the online version of Primer 3 software (Rozen and Skaletsky, 2000) to produce amplicons from 100 to 800 bp. The primers were synthesized by MWG Biotech AG (Ebersberg-Munich, Germany). One pair of primers were designed to amplify rose beta actin (Rh-ß-actin) cDNA based on the reported mRNA sequence of Rosa chinensis (Supplementary Table S1). Rh-ß-actin (a “househoulding” gene) was used as
The miniature rose cultivars ‘Vanilla’ (low ethylene-sensitivity) and ‘Lavender’ (high ethylene-sensitivity) were obtained from Rosen Kordes, W. Kordes’ Söhne Rosenschulen GmbH & Co KG, Sparrishoop, Germany. The 4 F1 genotypes used in this work (48, 131, 50 and 74) were selected as described by Ahmadi et al. (2009). Genotype 48 and 131 showed lower ethylene-sensitivity
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Fig. 1. Three flower stages for ‘Vanilla’ (left) and ‘Lavender’ (right): 1 Flowers in the opening phase. 2 Flowers fully open. 3 Flowers in initial phase of senescence.
an internal control for normalization of the other gene expressions. Corrections for differences in amplicon lengths for Rh-ß-actin and the other investigated transcripts were included in the normalizations. The exponential range was determined by carrying out PCR for 20–45 cycles using serial dilution of a fixed quantity of Rh-ßactin cDNA (Supplementary Fig. S1). As seen, the reaction appears to be in the exponential range for 30 or fewer cycles. Therefore, the RT-PCR reactions in this work was run for 30 cycles and with dilutions containing about as much Rh-ß-actin cDNA as the 10 fold diluted samples used in Supplementary Fig. S1. 2.4. RT-PCR Total RNA was reverse transcribed to cDNA as described above and PCR reactions were performed on the cDNA in a T3 thermocycler (Biometra, Germany) under the following conditions: an initial denaturation step at 95 ◦ C for 2 min, 30 cycles of denaturation at 95 ◦ C for 30 s, annealing at 50–62 ◦ C for 40 s, and extension at 72 ◦ C for 2 min, and a final extension at 72 ◦ C for 10 min. The PCR products were electrophoresed in a 1% agarose gel with 0.3 g mL−1 ethidium bromide in 1 × TAE buffer and visualized using a BioDocAnalyze UV transilluminator (Biometra, Germany). As a negative control for RT-PCR, a mixture of different randomly chosen RNA samples were subjected to the same reaction without reverse transcriptase. All RT-PCR experiments were repeated three times for each total RNA preparation. 2.5. Densitometric analysis of RT-PCR results Negative images of the agarose gels were analyzed to quantity the optical density of PCR bands from each sample using ImageQuant software (Amersham, GE Healthcare UK limited, Buckinghamshire, UK). The results were expressed as the relative density in comparison to the optical density of the background and normalized to the expression of the housekeeping gene for ß-actin, which is assumed to be equally expressed in most cells. 3. Results 3.1. Ethylene receptor genes (RhETR1-4) RhETR1, RhETR2 and RhETR3 transcripts were detected in all tissues tested for both ‘Vanilla’ and ‘Lavender’ (Fig. 2). Expression of RhETR4 was not detected, regardless of the type of tissue or growth stage (data not shown). In carpels, the amount of RhETR1 mRNA in ‘Vanilla’ was higher than for ‘Lavender’ at all developmental stages (Fig. 2). In petals, the amount of RhETR2 mRNA was higher for ‘Vanilla’ than for ‘Lavender’ at stage 2 and 3 and all three transcripts reached their maximum at stage 3 for ‘Vanilla’ but stage 2 for ‘Lavender’. In floral buds and in leaves the level of all three receptor transcripts was higher for ‘Vanilla’ than for ‘Lavender’. RhETR3 mRNA was higher for ‘Vanilla’ in buds and leaves whereas it was not expressed in Lavender in the same tissues (Fig. 2). The total amount of transcript for the three receptors (estimated as the sum
of the column heights) was higher for ‘Vanilla’ than for ‘Lavender’ in all cases except for stage 1 petals. For the selected F1 genotypes (Section 2.1), expression of RhETR1-4 was measured in petals of fully blooming flowers (stage 2, Fig. 1), floral buds and leaves from each of the selected F1 genotypes. As for the parents, no expression of RhETR4 was detected in the investigated tissues. In all cases except for genotype 48 buds the dominant transcripts are RhETR2 (Supplementary Fig. S2). In general, the differences between the genotypes of low and high ethylene sensitivity are small, and less conspicuous than some of the differences seen between ‘Vanilla’ and ‘Lavender’. 3.2. Genes for the signal transduction proteins RhCTR1 and RhCTR2 Expression of the RhCTR1 and RhCTR2 genes, which encode homologous protein kinases involved in ethylene signal transduction, were analyzed in ‘Vanilla’ and ‘Lavender’ (Fig. 3). RhCTR2 was significantly more expressed than RhCTR1 in all investigated tissues, except for ‘Lavender’ stage 3 carpels where both showed very low expression. The total amount of transcript for the two genes (estimated as the sum of the column heights) varied, with the exception just mentioned, from 0.33 (for ‘Vanilla’ stage 3 carpels) and 0.97 (for ‘Vanilla’ stage 3 petals). It was relatively constant for all 6 samples of petals. For the selected F1 plants the expression of RhCTR1 was significantly higher than, and that of RhCTR2 was, in general, at the same level as, the expressions found in ‘Vanilla’ and ‘Lavender’ (Supplementary Fig. S3). Accordingly, the sum of the expressions was higher than for the parent plants. 3.3. Genes for the transcription factors RhEIN3 and RhEIL Expression of the RhEIN3 and RhEIL genes, which encode homologous transcription factors involved in the ethylene signal pathway, were detected in all investigated tissues from ‘Vanilla’ and ‘Lavender’ (Fig. 4). In all cases, except for ‘Vanilla’ stage 1 carpels and ‘Lavender’ stage 3 petals, buds and leaves, the expression of RhEIL exceeded that of RhEIN3. In petals, the amount of both transcripts was maximal at stage 2 and 3 for ‘Vanilla’ but at stage 1 and 2 for ‘Lavender’. As for the parent plants, expression analysis of stage 2 petals, buds and leaves from the selected F1 plants detected both transcripts (Supplementary Fig. S4) and more RhEIL than RhEIN3 transcript. There is only little variation between expression in the 4 genotypes. For the petals, the total expressions are similar to that of stage 2 petals from the parent plants, and for leaves the total expressions are also similar. 3.4. Ethylene biosynthetic genes (RhACS1-5) Expression of the ethylene biosynthetic enzyme genes RhACS15 was analyzed in ‘Vanilla’ and ‘Lavender’. Whereas RhACS1 and RhACS2 transcripts were detected in all investigated tissues for both cultivars (Fig. 5), RhACS3-5 transcripts were not detected in any of
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Fig. 2. Expression patterns for RhETR1, RhETR2 and RhETR3 at three developmental stages of (carpels and petals), and in floral buds and leaves of ‘Vanilla’ and ‘Lavender’ as determined by RT-PCR. Each column represents the optical density of the PCR band divided by the optical density of the band for the internal standard Rhˇ-actin, with correction for difference in amplicon sizes. The values are given as means of densitometric units of three RT-PCR reactions; error bars represent standard error of the mean. The numbers at the top are the sum of the heights of the columns for RhETR1-3.
Fig. 3. Expression patterns for RhCTR1 and RhCTR2 at three developmental stages of (carpels and petals), and in floral buds and leaves of ‘Vanilla’ and ‘Lavender’ as determined by RT-PCR. Each column represents the optical density of the PCR band divided by the optical density of the band for the internal standard Rhˇ-actin, with correction for difference in amplicon sizes. The values are given as means of densitometric units of three RT-PCR reactions; error bars represent standard error of the mean. The numbers at the top are the sum of the heights of the columns for RhCTR1 and RhCTR2.
the investigated tissues (data not shown). In ‘Vanilla’ and ‘Lavender’ carpels and in ‘Vanilla’ petals, buds and leaves RhACS2 was more expressed than RhACS1, whereas the opposite was the case for ‘Lavender’ petals (except for stage 1), buds and leaves. The sum of the two transcript levels is in all cases larger for ‘Vanilla’ than for ‘Lavender’. As for the parent plants, expression analysis of stage 2 petals, buds and leaves from the selected F1 plants detected RhACS1 and RhACS2 transcripts (Supplementary Fig. S5), but no RhACS3, RhACS4 and RhACS5 transcripts (data not shown), in the investigated tissues. The expression of RhACS1 was always higher than the RhACS2 expression in petal tissue, as for ‘Lavender’ but not for ‘Vanilla’ (Fig. 5), regardless of sensitivity to ethylene (Supplementary Fig. S5). In addition, the sum of the two transcript levels in petals is larger than for both ‘Lavender’ and ‘Vanilla’ for all 4 genotypes. For leaves and buds the patterns were more variable, but without conspicuous correlation with ethylene sensitivity.
4. Discussion To clarify the reasons for the significant differences in postharvest characteristics of miniature potted roses, mRNA expression patterns of genes involved in ethylene biosynthesis, perception and signal transduction pathways were investigated and compared for the cultivars ‘Vanilla’ and ‘Lavender’, which show low and high ethylene sensitivity, respectively, and plants selected among their F1 offspring for low and high ethylene sensitivity (Section 2.1). Based on the current model of ethylene signal transduction (briefly described in the Introduction), and the experimental evidence behind it, it may be expected that plants with increased levels of receptor protein, increased CTR1 levels, decreased EIN2 levels, or decreased EIN3/EIL levels should show decreased ethylene sensitivity. We investigated the transcript levels for the cloned Rosa hybrida genes for ethylene signal transduction proteins in the above-mentioned cultivars to look for a possible correlation
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Fig. 4. Expression patterns for RhEIN3 and RhEIL at three developmental stages of (carpels and petals), and in floral buds and leaves of ‘Vanilla’ and ‘Lavender’ as determined by RT-PCR. Each column represents the optical density of the PCR band divided by the optical density of the band for the internal standard Rhˇ-actin, with correction for difference in amplicon sizes. The values are given as means of densitometric units of three RT-PCR reactions; error bars represent standard error of the mean. The numbers at the top are the sum of the heights of the columns for RhEIN3 and RhEIL.
Fig. 5. Expression patterns for RhACS1 and RhACS2 at three developmental stages of (carpels and petals), and in floral buds and leaves of ‘Vanilla’ and ‘Lavender’ as determined by RT-PCR. Each column represents the optical density of the PCR band divided by the optical density of the band for the internal standard Rhˇ-actin, with correction for difference in amplicon sizes. The values are given as means of densitometric units of three RT-PCR reactions; error bars represent standard error of the mean. The numbers at the top are the sum of the heights of the columns for RhACS1 and RhACS2.
between transcript levels and ethylene sensitivity that could confirm (or disagree with) these expectations. RhEIN2 is not yet cloned and was therefore not investigated. Müller et al. (2000a) reported that the expression of RhETR1 in petals from the cultivar ‘Bronze’ with short flower life was clearly higher than for the long-lasting ‘Vanilla’, in contrast to what could be expected from the ethylene signal transduction model. We found, however, that the amount of RhETR1 mRNA in ‘Vanilla’ was higher than for the short-lasting cultivar ‘Lavender’ at all developmental stages in carpels, at stage 3 in petals, and in buds (Fig. 2). In floral buds and in leaves the levels of all three receptor transcripts were higher for ‘Vanilla’ than for ‘Lavender’ (Fig. 2). In addition, the total amount of transcript for RhETR1-3 was higher for ‘Vanilla’ than for ‘Lavender’ in all cases except for stage 1 petals and leaves. These findings are what would be expected from the signal transduction pathway. The expression of RhETR1 in F1 progeny plants of ‘Vanilla’ and ‘Lavender’ was evaluated for petals, floral buds, and
leaves (Supplementary Fig. S2). RhETR1 was expressed in all tissues, and no clear differences were seen between the long-lasting genotypes 48 and 131 and the short-lasting genotypes 50 and 74. The long-lasting genotypes, however, had significantly lower total receptor expression in buds and leaves (but not in petals) than the short-lasting genotypes, which is not what could be expected from the model of ethylene signal transduction. For ‘Vanilla’, in contrast, the total receptor expression in buds is larger than for ‘Lavender’ buds (Fig. 2). In petals, the amount of RhETR2 mRNA was higher for ‘Vanilla’ than for ‘Lavender’ at stage 2 and 3 and all three transcripts reached their maximum at stage 3 for ‘Vanilla’ but stage 2 for ‘Lavender’ (Fig. 2). RhETR2 expression was higher than RhETR1 expression for ‘Vanilla’ petals, ‘Lavender’ stage 3 petals and ‘Lavender’ buds, whereas the opposite was the case for the other analyzed tissues (Fig. 2). These results for petal RhETR2 transcripts are not in agreement with observations by Müller et al. (2000b), who found that
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the expression of RhETR2 in petals of the two cultivars ‘Bronze’ and ‘Vanilla’ is constitutive during flower senescence, and that the transcript abundance was higher in ‘Bronze’ (more ethylene sensitive) than in ‘Vanilla’ (less ethylene sensitive) at all three stages of development. RhETR2 was expressed in all analyzed tissues of the selected F1 plants (Fig. 2). Remarkably, RhETR2 was significantly more expressed than RhETR1 or RhETR3, except for genotype 48 buds. The expression of RhETR3 was lower than 0.2 (in many cases lower than 0.1) in all analyzed ‘Vanilla’ and ‘Lavender’ tissues, except for ‘Vanilla’ leaves (Fig. 2). The same was the case for the F1 genotypes (Supplementary Fig. S2). Müller et al. (2000b) reported that the level of mRNA for RhETR3 in petals of ‘Bronze’ was clearly higher than for ‘Vanilla’. In contrast to this observation, the RhETR3 mRNA level for stage 3 ‘Vanilla’ petals is higher than the level for stage 3 ‘Lavender’ petals and it was not expressed in buds and leaves of ‘Lavender’ (Fig. 2). Since there are some differences in RhETR3 expression between ‘Vanilla’ and ‘Lavender’, further research is required to clarify the relation between the RhETR3 transcript level and ethylene sensitivity. There was no detectable expression of RhETR4 in any of the analyzed ‘Lavender’ and ‘Vanilla’ or F1 plant tissues. In accordance with these observations, Xue et al. (2008) did not detect RhETR4 expression in gynoecia, petals, stamens, sepals, and receptacles of the rose cultivar ‘Samantha’. Müller et al. (2000b) isolated a 547 bp RhETR4 sequence. The amino acid sequence deduced from this internal fragment of RhETR4 is 79% identical to that for RhETR1. However, the authors did not investigate the RhETR4 expression. Expression of the RhCTR1 and RhCTR2 genes, which encode protein kinases that inhibit EIN2 when ethylene is not bound to the receptors, were analyzed. RhCTR2 was significantly more expressed than RhCTR1 in all investigated tissues, except for ‘Lavender’ stage 3 carpels where both showed very low expression. It should also be noted that for petals there is an increase in total expression when petals develop from stage 2 to stage 3 for ‘Vanilla’ but a decrease for ‘Lavender’ (Fig. 3). Hajizadeh et al. (2011) obtained similar results as they observed that expression of RhCTR1 increased during flower opening and that expression of RhCTR1 was higher in a cultivar with long vase life than in one with short vase life. Müller et al. (2002, 2003) observed no expression differences of RhCTR1 and RhCTR2 between the long-lasting ‘Vanilla’ and the short-lasting ‘Bronze’ cultivar, but found an increase of RhCTR1 expression in petals during flower development. The higher level of total expression in ‘Vanilla’ than in ‘Lavender’ stage 3 carpels and petals is in accordance with the standard model for the ethylene signal transduction pathway, since that predicts that higher CTR expression should result in less ethylene sensitive plants. The RhEIN3 and RhEIL genes encode transcription factors that, in the presence of ethylene, activates the transcriptional response to ethylene. RhEIN3 and RhEIL expression was detected in all investigated tissues (Figs. 4 and S4). In all cases, except for a few tissues from the parent plants, the expression of RhEIL exceeded that of RhEIN3. For the F1 plants, there was only little variation between expression in the 4 genotypes, and the total expressions in petals were similar to that of stage 2 petals from the parent plants. For the 3 stages of ‘Vanilla’ and ‘Lavender’ petals the expression levels are relatively constant. In accordance with these observations, Müller et al. (2003) reported constitutive and stable expression of RhEIN3 in petals of ‘Vanilla’ and ‘Bronze’ cultivars during flower senescence, and Ma et al. (2006) reported constant expression of Rh-EIN3-1 (here termed RhEIN3) and Rh-EIN3-2 (here termed RhEIL) in cut ‘Samantha’ roses. Consequently, if there are differences in RhEIN3 and RhEIL activity in cultivars showing different ethylene sensitivity, they are probably mostly due to posttranscriptional control such as control of protein stability (Cho and Yoo 2015).
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High susceptibility to ethylene may possibly be due to induction of increased activity of ethylene biosynthetic enzymes by exposure to external ethylene. In higher plants, ethylene biosynthesis has been well characterized (Yang and Hoffman, 1984), and many studies have been performed on regulation of the enzymes involved (Xu and Zhang 2015). ACC synthase (ACS) and ACC oxidase (ACO), the two key enzymes in the ethylene biosynthetic pathway, are regulated both transcriptionally and post transcriptionally (Xu and Zhang 2015). Thain et al. (2004) showed, eg., that expression of ACC synthase genes in Arabidopsis is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. Since ACS is generally considered the rate-limiting enzyme of ethylene biosynthesis we decided to study the 5 ACC synthase genes for which rose sequences were available. The RT-PCR result showed that only RhACS1 and RhACS2 were expressed in the investigated tissues of ‘Vanilla’, ‘Lavender’ and the selected F1 plants, while RhACS3, RhACS4 and RhACS5 expression could not be detected. The sum of the two transcript levels is in all cases larger for ‘Vanilla’ than for ‘Lavender’ (Fig. 5). This is contrary to what could be expected if it is assumed that transcript level correlates with enzyme activity and that ethylene production in the tissues contributes to a lower ethylene sensitivity. For the F1 plants, expression of RhACS1 was always higher than the RhACS2 expression in petal tissue (Supplementary Fig. S5). In addition, the sum of the two transcript levels in petals is larger than for both ‘Lavender’ and ‘Vanilla’ for all 4 genotypes. Again, there is no obvious correlation between transcript levels and ethylene sensitivity. Müller et al. (2000a) found that the ACC synthase transcript (detected by northern-blotting and use of a Pelargonium ACC synthase probe) increased during flower senescence in ‘Vanilla’ but remained constant at a low level in ‘Bronze’. That the level was higher in ‘Vanilla’ than in ‘Bronze’ agrees with our finding of a higher total expression in ‘Vanilla’ than in ‘Lavender’ petals (Fig. 5). Liu et al. (2013) reported RhACS1-3 expression in cut ‘Samantha’ roses throughout the dehydration period. They also reported that RhACS4 and RhACS5 expression could hardly be detected. A recent study of Khan et al. (2015) showed that the promoter activities of RhACS1 and RhACS2 in ‘Samantha’ were strong during the development from young seedlings to mature flowering plants in various investigated organs, including hypocotyls, cotyledons, leaves, roots and lateral roots. Except for the RhACS3 expression, these results agree with those obtained for ‘Vanilla’, ‘Lavender’ and their F1 progeny. From the observations in the present work it is not possible to find a clear correlation between transcript levels of the analyzed genes and the ethylene-sensitivity of the studied rose cultivars. A lack of clear correlation between transcription of ethylene signal transduction genes and ethylene sensitivity was also noted by Ahmadi et al. (2009), but it was established for more genes in this study. These observations indicate that much of the variation seen for transcript levels may be due to random factors (such as variation in the genotypes and epigenetic state of the analyzed plants) that are not important for ethylene sensitivity, and that other genes or factors could more tightly regulate the sensitivity to ethylene. In accordance with this, recent molecular investigations of the ethylene signal transduction pathway have revealed other processes, such as proteasomal degradation and MAP kinase cascades, that are involved in the signal processes (Cho and Yoo 2015). It is therefore likely that transcription of other genes involved in ethylene signal transduction, posttranscriptional or posttranslational control, or crosstalk with other signal transduction pathways may influence the degree of ethylene sensitivity of miniature roses.
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Acknowledgements The experimental work was partly supported by a PhD grant from the German Academic Exchange Service (DAAD) (Ref.: 412, PKZ: A/03/33925). The authors would like to thank Prof. em. Bjarne M. Stummann (University of Copenhagen, Denmark) for his valuable advices and critical review of the manuscript. Authors would also like to thank technical and scientific staff: Dr. Heiko Mibus, Mrs. Annette Steding and Mr. Herbert Geyer, from the Section of Floriculture at Leibniz University of Hannover for fruitful discussions and technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2016. 12.029. References Ahmadi, N., Mibus, H., Serek, M., 2009. Characterization of ethylene-induced organ abscission in F1 breeding lines of miniature roses (Rosa hybrida L.). Postharvest Biol. Technol. 52, 260–266. Andersen, L., Williams, M.H., Serek, M., 2004. Reduced water availability improves drought tolerance of potted miniature roses; Is the ethylene pathway involved? J. Hortic. Sci. Biotech. 79, 1–13. Buanong, M., Mibus, H., Sisler, E.C., Serek, M., 2005. Efficacy of new inhibitors of ethylene perception in improvement of display quality of miniature potted roses (Rosa hybrida L.). Plant Growth Regul. 47, 29–38. Cancel, J.D., Larsen, P.B., 2002. Loss-of-Function mutations in the ethylene ReceptorETR1 cause enhanced sensitivity and exaggerated response to ethylene in arabidopsis. Plant Physiol. 129, 1557–1567. Cho, Y.H., Yoo, S.D., 2015. Novel connections and gaps in ethylene signaling from the ER membrane to the nucleus. Front. Plant Sci. 5, 1–7. Ferrante, A., Trivellini, A., Scuderi, D., Romano, D., Vernieri, P., 2015. Post-production physiology and handling of ornamental potted plants. Postharvest Biol. Technol. 100, 99–108. FloraHolland, 2016. Flora Holland: Our Facts & Figures Annual Report 2015. Koninklijke Coöperatieve Bloemenveiling Flora Holland U.A.16. Gallie, D.R., 2015. Ethylene receptors in plants-why so much complexity? F1000Prime Reports 7, 1–12. Hajizadeh, H., Razavi, K., Mostofi, Y., Cacco, G., Mousavi, A., Zamani, Z., Stevanato, P., 2011. Expression of genes encoding protein kinases during flower opening in two cut rose cultivars with different longevity. Iran. J. Biotechnol. 9, 230–233. Ju, C., Yoon, G.M., Shemansky, J.M., Lin, D.Y., Ying, Z.I., Chang, J., Garrett, W.M., Kessenbrock, M., Groth, G., Tucker, M.L., Cooper, B., 2012. CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proc. Natl Acad. Sci 109, 19486–19491. Khan, M.A., Meng, Y., Liu, D., Tang, H., Lü, S., Imtiaz, M., Jiang, G., Lü, P., Ji, Y., Gao, J., Ma, N., 2015. Responses of rose RhACS1 and RhACS2 promoters to abiotic stresses in transgenic Arabidopsis thaliana. Plant Cell Rep. 34, 795–804. Liu, D., Liu, X., Meng, Y., Sun, C., Tang, H., Jiang, Y., Khan, M.A., Xue, J., Ma, N., Gao, J., 2013. An organ-specific role for ethylene in rose petal expansion during dehydration and rehydration. J. Exp. Bot. 64, 2333–2344. Müller, R., Andersen, A.S., Serek, M., 1998. Differences in display life of miniature potted roses (Rosa hybrida L.). Sci. Hortic. 76, 59–71. Müller, R., Lind-Iversen, S., Stummann, B.M., Serek, M., 2000a. Expression of genes for ethylene biosynthetic enzymes and an ethylene receptor in senescing flowers of miniature roses. J. Hortic. Sci. Biotech. 75, 12–18.
Müller, R., Stummann, B.M., Serek, M., 2000b. Characterization of an ethylene receptor family with differential expression in rose (Rosa hybrida L.) flowers. Plant Cell Rep. 19, 1232–1239. Müller, R., Stummann, B.M., Sisler, E.C., Serek, M., 2001. Cultivar differences in regulation of ethylene production in miniature rose flowers (Rosa hybrida L.). Gartenbauwissenschaft 66, 34–38. Müller, R., Owen, C.A., Xue, Z.T., Welander, M., Stummann, B.M., 2002. Characterization of two CTRülike protein kinases in Rosa hybrida and their expression during flower senescence and in response to ethylene. J. Exp. Bot. 53, 1223–1225. Müller, R., Owen, C., Xue, Z.T., Welander, M., Stummann, B.M., 2003. The transcription factor EIN3 is constitutively expressed in miniature roses with differences in postharvest life. J. Hortic. Sci. Biotech. 78, 10–14. Ma, N., Tan, H., Liu, X., Xue, J., Li, Y., Gao, J., 2006. Transcriptional regulation of ethylene receptor and CTR genes involved in ethylene-induced flower opening in cut rose (Rosa hybrida) cv. Samantha. J. Exp. Bot. 57, 2763–2773. Merchante, C., Alonso, J.M., Stepanova, A.N., 2013. Ethylene signaling: simple ligand: complex regulation. Curr. Opin. Plant. Biol. 16, 554–560. Mibus, H., Serek, M., 2004. Easy PCR method to isolate unknown ACC synthase genes in ornamental plant species. Acta Hortic. 682, 307–312. Pemberton, H.B., Kelly, J.W., Ferare, J., 2003. Pot rose production. In: Roberts, A., Debener, T., Gudin, S. (Eds.), Encyclopedia of Rose Science. Elsevier Science, Oxford, pp. 587–593. Rozen, S., Skaletsky, H., 2000. Primer3 on the WWW for general users and for biologist programmers. In: Krawetz, S., Misener, S. (Eds.), Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, pp. 365–386. Serek, M., Woltering, E.J., Sisler, E.C., Frello, S., Sriskandarajah, S., 2006. Controlling ethylene responses in flowers at the receptor level. Biotechnol. Adv. 24, 368–381. Serek, M., 1993. Ethephon and silver thiosulfate affect postharvest characteristics of Rosa hybrida ‘Victory Parade’. HortScience 28, 199–200. Shakeel, S.N., Wang, X., Binder, B.M., Schaller, G.E., 2013. Mechanisms of signal transduction by ethylene: overlapping and non-overlapping signalling roles in a receptor family. AoB Plants 5, plt010. Shakeel, S.N., Gao, Z., Amir, M., Chen, Y.F., Rai, M.I., Haq, N.U., Schaller, G.E., 2015. Ethylene regulates levels of ethylene receptor/CTR1 signaling complexes in Arabidopsis thaliana. J. Biol. Chem. 290, 12415–12424. Tan, H., Liu, X., Ma, N., Xue, J., Lu, W., Bai, J., Gao, J., 2006. Ethylene-influenced flower opening and expression of genes encoding ETRs CTRs, and EIN3 s in two cut rose cultivars. Postharvest Biol. Technol. 40, 97–105. Thain, S.C., Vandenbussche, F., Laarhoven, L.J., Dowson-Day, M.J., Wang, Z.Y., Tobin, E.M., Harren, F.J., Millar, A.J., Van Der Straeten, D., 2004. Circadian rhythms of ethylene emission in Arabidopsis. Plant Physiol. 136, 3751–3761. Tieman, D.M., Taylor, M.G., Ciardi, J.A., Klee, H.J., 2000. The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family. Proc. Natl. Acad. Sci. U. S. A. 97, 5663–5668. Wang, D., Fan, J., Ranu, R.S., 2004. Cloning and expression of 1-aminocyclopropane-1-carboxylate synthase cDNA from rosa (Rosa x hybrida). Plant Cell Rep. 22, 422–429. Wilkins, T.A., Smart, L.B., 1996. Isolation of RNA from plant tissue. In: Krieg, P.A. (Ed.), A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis. Wiley-Liss, Inc., New York, NY, pp. 21–42. Wuriyanghan, H., Zhang, B., Cao, W.H., Ma, B., Lei, G., Liu, Y.F., Wei, W., Wu, H.J., Chen, L.J., Chen, H.W., Cao, Y.R., 2009. The ethylene receptor ETR2 delays floral transition and affects starch accumulation in rice. Plant Cell 21, 1473–1494. Xu, J., Zhang, S., 2015. Ethylene biosynthesis and regulation in plants. In: Wen, C.-K. (Ed.), Ethylene in Plants. Springer, Netherlands, pp. 1–25. Xue, J., Li, Y., Tan, H., Yang, F., Ma, N., Gao, J., 2008. Expression of ethylene biosynthetic and receptor genes in rose floral tissues during ethylene-enhanced flower opening. J. Exp. Bot. 59, 2161–2169. Yang, S.F., Hoffman, N.E., 1984. Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant. Physiol. 35, 155–189.
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Expression analysis by RT-PCR of genes involved in ethylene synthesis and signal transduction in miniature roses Mohammad Al-Salem, Margrethe Serek ∗ Leibniz University of Hannover, Faculty of Natural Sciences, Institute of Horticulture Production Systems, Floriculture, Herrenhaeuser Str. 2, D-30419 Hanover, Germany
a r t i c l e
i n f o
Article history: Received 30 September 2016 Received in revised form 27 December 2016 Accepted 29 December 2016 Keywords: Ethylene receptor Ethylene sensitivity Gene expression Postharvest Rosa hybrida L. Signal transduction
a b s t r a c t Ethylene is one of many factors that affect the quality, appearance and longevity of miniature roses. In this study RT-PCR is used to compare the expression of ethylene biosynthetic genes and ethylene signal transduction genes at different stages of flower development in the two cultivars ‘Vanilla’ and ‘Lavender’, which show low and high ethylene-sensitivity, respectively, and their F1 offspring. The genes for the ethylene receptors RhETR1, RhETR2 and RhETR3, the genes for the receptor-associated signaling proteins RhCTR1 and RhCTR2, the genes for the transcription factors RhEIN3 and RhEIL, and the genes for the ACC synthases RhACS1 and RhACS2 each had an expression pattern that varied between the tested plants and tissues, but could not be correlated with the ethylene sensitivity of the plants. RhETR1, RhETR2, RhETR3 and RhEIN3 were, e.g., expressed more in ‘Vanilla’ than in ‘Lavender’ in most, but not all, of the investigated tissues, but were in general not expressed more in progeny with low sensitivity than in progeny with high sensitivity. No expression was detected for the 4 other genes that were investigated, i.e. genes for RhETR4, RhACS3, RhACS4 and RhACS5. It is concluded that the precise transcriptional activities of the tested genes do not appear to be crucial in determining the ethylene sensitivity of miniature roses. It therefore appears likely that transcription of other genes involved in ethylene signal transduction, posttranscriptional or posttranslational control, or crosstalk with other signal transduction pathways may be important for the degree of ethylene sensitivity of miniature roses. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Miniature rose, Rosa hybrida L., is one of the most economically important ornamental plants for indoor use. Miniature potted roses have become very popular in the last two decades due to their availability in a wide range of colors, sizes, growth habits, and to improved postharvest performance. The annual world production is estimated to be more than 100 million plants, and Denmark is the largest producer with nearly 50% of the world’s production (Pemberton et al., 2003). The number of miniature rose plants sold in 2015 at Flora Holland, the world largest flower auction, reached 47 million, generating revenues of EUR 57 million (FloraHolland, 2016). Ethylene is one of the most important factors affecting the quality, appearance and longevity of many ornamentals (Serek et al., 2006; Ferrante et al., 2015). In miniature roses, ethylene can cause unwanted effects such as premature and accelerated wilting and
∗ Corresponding author. E-mail address: [email protected] (M. Serek). http://dx.doi.org/10.1016/j.scienta.2016.12.029 0304-4238/© 2016 Elsevier B.V. All rights reserved.
abscission of leaves, floral buds, petals and flowers, as well as leaf yellowing or discoloration (Serek 1993; Andersen et al., 2004). The display life of some commercially grown miniature potted rose cultivars was compared by Müller et al. (1998), and it was found that it varied considerably, at least partly due to differences in endogenous ethylene production and sensitivity to exogenous ethylene. In higher plants, the mechanism of ethylene biosynthesis has been well investigated (Yang and Hoffman, 1984; Xu and Zhang 2015). Ethylene signal transduction has also been investigated extensively, resulting in a model encompassing the following wellestablished aspects (Merchante et al., 2013; Cho and Yoo 2015; Gallie 2015; Shakeel et al., 2015). In the absence of ethylene, the ethylene receptors, located in the ER membrane, activate CTR1 (and its homologs) which is associated with the receptors and, when activated, phosphorylates and inhibits EIN2 (a membrane protein also located in the ER membrane). In the presence of ethylene, CTR1 is “switched off”, and EIN2 becomes dephosphorylated, leading to release of its C-terminal domain (Ju et al., 2012). This domain translocates to the nucleus, where it blocks degradation of the transcription factors EIN3/EIL1 and thus activates the transcriptional response to ethylene. Other, less well-characterized proteins,
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also are involved in the signal transduction pathway (Cho and Yoo 2015). There are several ethylene receptors (5 in Arabidopsis) with partly overlapping function (Shakeel et al., 2013; Gallie 2015). From this model, it may be expected that increased amounts of receptor and CTR1 proteins would lead to less ethylene-sensitive plants, whereas increased amounts of EIN2 and/or EIN3/EIL1 proteins would lead to more ethylene-sensitive plants. In attempts to clarify the molecular causes for the differences in flower longevity among miniature rose cultivars, the expression of transcripts for ethylene biosynthetic genes, ethylene receptor genes and other ethylene signal transduction genes have been investigated (Müller et al., 2000a, 2000b, 2001, 2002, 2003; Xue et al., 2008; Ahmadi et al., 2009). For miniature roses, sequences from 4 ethylene receptor genes, namely RhETR1, RhETR2, RhETR3, and RhETR4, have been isolated by Müller et al. (2000a, b), and a fifth gene, RhETR5, was identified by Tan et al. (2006). Mibus and Serek (2004) isolated sequences from 5 ACC synthase genes and Wang et al. (2004) isolated 7 ACC synthase cDNAs. Two genes for ethylene receptor-associated kinases have been identified in miniature roses, namely RhCTR1 and RhCTR2 (Müller et al., 2002). In 2003 Müller et al. identified a gene with high homology to the Arabidopsis gene for the transcription factor EIN3 (also involved in ethylene signal transduction). This gene is in the present work referred to as RhEIL (EIN3-like). In 2006 Tan et al. identified another EIN3-like sequence (here termed RhEIN3) in cut roses. As reported by Müller et al. (1998) ‘Vanilla’ is a cultivar with low sensitivity to ethylene. This cultivar has later been crossed to the highly sensitive cultivar ‘Lavender’ (Buanong et al., 2005; Ahmadi et al., 2009). Some F1 plants (genotypes 50 and 74) were more sensitive to ethylene than ‘Lavender’ while some (genotypes 48 and131) were less sensitive to ethylene than ‘Vanilla’ as judged from the rate of bud or leaf abscission during 15 days of exposure to ethylene. The expression of RhETR1, RhETR3, RhCTR1 and RhCTR2 transcripts in pedicels and petioles were tested in these F1 plants and the parent cultivars after 3 days of ethylene exposure, but no correlation to ethylene sensitivity was found (Ahmadi et al., 2009). Observations on ethylene sensitivity and receptor expression that are in accordance with the current model of ethylene signaling, have been reported by Tieman et al. (2000), who found that transgenic tomatoes with reduced LeETR4 gene expression show high ethylene sensitivity, by Wuriyanghan et al. (2009), who reported that overexpression of the ethylene receptor gene OsETR2 in rice reduces ethylene sensitivity, and by Cancel and Larsen (2002), who found that mutational loss of the ethylene receptor ETR1 in Arabidopsis results in a significant increase in sensitivity to ethylene. In the present study, different tissues from two cultivars ‘Vanilla’ and ‘Lavender’, which show low and high ethylene-sensitivity, respectively, and their F1 offspring were tested by RT-PCR at different developmental stages for transcripts of 8 ethylene transduction genes (RhETR1-4, RhCTR1-2, RhEIN3 and RhEIL) and 5 ACC synthase genes (RhACS1-5) in order to correlate ethylene sensitivity with expression of one or more of these genes and thus explain the differences between postharvest life of the tested cultivars.
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than ‘Vanilla’ and genotype 50 and 74 showed higher ethylenesensitivity than ‘Lavender’. The plants were grown in greenhouses under the following conditions: 22 ◦ C day/20 ◦ C night temperatures and 60–85% relative humidity with 350 mol m−2 s−1 of light from SON-T lamps (Osram, 400 W, Philips, Eindhoven, The Netherlands) with a supplement of natural daylight over a 16 h photoperiod. Total RNA was isolated from selected plant tissues and stages of development. Expression patterns for the genes investigated were determined in carpels and petals from stage 1–3 flowers (Fig. 1), floral buds, and leaves of ‘Vanilla’ and ‘Lavender’, whereas, for the F1 plants, it was investigated in petals of the fully blooming stage (stage 2) flower floral buds and leaves. 2.2. Total RNA extraction and cDNA synthesis A modified hot borate method (Wilkins and Smart, 1996) was used to isolate a high yield of total RNA. Two g of finely ground, frozen plant material of each kind were mixed with 8 mL of the 80–90 ◦ C hot extraction buffer (1% SDS, 1% sodium deoxycholic acid, 5 mM EDTA, 0.2 M Borax (Sodium borate decahydrate)) in 50 mL tubes. PVP (2%), DTT (1%), and Np-40 (1%) had been added to the extraction buffer just before heating. 110 L of Proteinase K (20 mg/mL) was added to each tube. The tubes were incubated at 42 ◦ C for 90 min in a water bath, and were vortexed each 10 min. Then the tubes were removed from water bath and 2 M KCl was added to a final concentration of 160 mM KCl. The tubes were shaken vigorously and incubated 1 h on ice. After that the tubes were centrifuged at 10000g for 20 min at 4 ◦ C. After centrifugation, the aqueous phase was transferred to 20 mL tubes and 8 M LiCl was added to a final concentration of 2 M LiCl. The tubes were placed in a refrigerator (4 ◦ C) overnight. The following day, RNA was precipitated by centrifugation at 14000g at 4 ◦ C for 20 min. The supernatants were discarded and the pellets were washed in 4 mL 2 M LiCl and suspended in 2 mL 10 mM Tris-HCl (pH 7.5) by gentle vortexing. Insoluble materials were removed from the RNA pellet by centrifugation at 10000g for 10 min at 4 ◦ C. After several steps of washing and centrifuging, the pellet was dissolved in 300 L DEPC-treated water. DNase I treatment was performed as described by manufacture (Fermentas GmbH, Germany) to degrade contaminating genomic DNA. First strand complementary DNA (cDNA) was prepared using M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant kit (Promega, USA). One g of total RNA was mixed with 1 g of the Oligo dT (23) primer in a total volume of 7 L. The mixture was heated to 70 ◦ C for 5 min using a T3 thermocycler (Biometra, Germany) and quickly cooled on ice for 5 min. Then the following components were added to the annealed primer in the order: M-MLV RT Reaction buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 , 10 mM DTT) (Promega, USA), 0.5 mM dNTP, 100 units M-MLV RT (H-) (Promega, USA) and 20 units of RNase out (Fermentas GmbH, Germany) in a total volume of 12.5 L. This reaction mixture was incubated for 10 min at 40 ◦ C followed by 150 min at 50 ◦ C and finally by 15 min at 70 ◦ C for deactivation of the reverse transcriptase.
2. Materials and methods
2.3. PCR primer design and optimization of number of PCR cycles
2.1. Plant material
The primer pairs used in this study are summarized in Supplementary Table S1. The primers were designed by use of the online version of Primer 3 software (Rozen and Skaletsky, 2000) to produce amplicons from 100 to 800 bp. The primers were synthesized by MWG Biotech AG (Ebersberg-Munich, Germany). One pair of primers were designed to amplify rose beta actin (Rh-ß-actin) cDNA based on the reported mRNA sequence of Rosa chinensis (Supplementary Table S1). Rh-ß-actin (a “househoulding” gene) was used as
The miniature rose cultivars ‘Vanilla’ (low ethylene-sensitivity) and ‘Lavender’ (high ethylene-sensitivity) were obtained from Rosen Kordes, W. Kordes’ Söhne Rosenschulen GmbH & Co KG, Sparrishoop, Germany. The 4 F1 genotypes used in this work (48, 131, 50 and 74) were selected as described by Ahmadi et al. (2009). Genotype 48 and 131 showed lower ethylene-sensitivity
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Fig. 1. Three flower stages for ‘Vanilla’ (left) and ‘Lavender’ (right): 1 Flowers in the opening phase. 2 Flowers fully open. 3 Flowers in initial phase of senescence.
an internal control for normalization of the other gene expressions. Corrections for differences in amplicon lengths for Rh-ß-actin and the other investigated transcripts were included in the normalizations. The exponential range was determined by carrying out PCR for 20–45 cycles using serial dilution of a fixed quantity of Rh-ßactin cDNA (Supplementary Fig. S1). As seen, the reaction appears to be in the exponential range for 30 or fewer cycles. Therefore, the RT-PCR reactions in this work was run for 30 cycles and with dilutions containing about as much Rh-ß-actin cDNA as the 10 fold diluted samples used in Supplementary Fig. S1. 2.4. RT-PCR Total RNA was reverse transcribed to cDNA as described above and PCR reactions were performed on the cDNA in a T3 thermocycler (Biometra, Germany) under the following conditions: an initial denaturation step at 95 ◦ C for 2 min, 30 cycles of denaturation at 95 ◦ C for 30 s, annealing at 50–62 ◦ C for 40 s, and extension at 72 ◦ C for 2 min, and a final extension at 72 ◦ C for 10 min. The PCR products were electrophoresed in a 1% agarose gel with 0.3 g mL−1 ethidium bromide in 1 × TAE buffer and visualized using a BioDocAnalyze UV transilluminator (Biometra, Germany). As a negative control for RT-PCR, a mixture of different randomly chosen RNA samples were subjected to the same reaction without reverse transcriptase. All RT-PCR experiments were repeated three times for each total RNA preparation. 2.5. Densitometric analysis of RT-PCR results Negative images of the agarose gels were analyzed to quantity the optical density of PCR bands from each sample using ImageQuant software (Amersham, GE Healthcare UK limited, Buckinghamshire, UK). The results were expressed as the relative density in comparison to the optical density of the background and normalized to the expression of the housekeeping gene for ß-actin, which is assumed to be equally expressed in most cells. 3. Results 3.1. Ethylene receptor genes (RhETR1-4) RhETR1, RhETR2 and RhETR3 transcripts were detected in all tissues tested for both ‘Vanilla’ and ‘Lavender’ (Fig. 2). Expression of RhETR4 was not detected, regardless of the type of tissue or growth stage (data not shown). In carpels, the amount of RhETR1 mRNA in ‘Vanilla’ was higher than for ‘Lavender’ at all developmental stages (Fig. 2). In petals, the amount of RhETR2 mRNA was higher for ‘Vanilla’ than for ‘Lavender’ at stage 2 and 3 and all three transcripts reached their maximum at stage 3 for ‘Vanilla’ but stage 2 for ‘Lavender’. In floral buds and in leaves the level of all three receptor transcripts was higher for ‘Vanilla’ than for ‘Lavender’. RhETR3 mRNA was higher for ‘Vanilla’ in buds and leaves whereas it was not expressed in Lavender in the same tissues (Fig. 2). The total amount of transcript for the three receptors (estimated as the sum
of the column heights) was higher for ‘Vanilla’ than for ‘Lavender’ in all cases except for stage 1 petals. For the selected F1 genotypes (Section 2.1), expression of RhETR1-4 was measured in petals of fully blooming flowers (stage 2, Fig. 1), floral buds and leaves from each of the selected F1 genotypes. As for the parents, no expression of RhETR4 was detected in the investigated tissues. In all cases except for genotype 48 buds the dominant transcripts are RhETR2 (Supplementary Fig. S2). In general, the differences between the genotypes of low and high ethylene sensitivity are small, and less conspicuous than some of the differences seen between ‘Vanilla’ and ‘Lavender’. 3.2. Genes for the signal transduction proteins RhCTR1 and RhCTR2 Expression of the RhCTR1 and RhCTR2 genes, which encode homologous protein kinases involved in ethylene signal transduction, were analyzed in ‘Vanilla’ and ‘Lavender’ (Fig. 3). RhCTR2 was significantly more expressed than RhCTR1 in all investigated tissues, except for ‘Lavender’ stage 3 carpels where both showed very low expression. The total amount of transcript for the two genes (estimated as the sum of the column heights) varied, with the exception just mentioned, from 0.33 (for ‘Vanilla’ stage 3 carpels) and 0.97 (for ‘Vanilla’ stage 3 petals). It was relatively constant for all 6 samples of petals. For the selected F1 plants the expression of RhCTR1 was significantly higher than, and that of RhCTR2 was, in general, at the same level as, the expressions found in ‘Vanilla’ and ‘Lavender’ (Supplementary Fig. S3). Accordingly, the sum of the expressions was higher than for the parent plants. 3.3. Genes for the transcription factors RhEIN3 and RhEIL Expression of the RhEIN3 and RhEIL genes, which encode homologous transcription factors involved in the ethylene signal pathway, were detected in all investigated tissues from ‘Vanilla’ and ‘Lavender’ (Fig. 4). In all cases, except for ‘Vanilla’ stage 1 carpels and ‘Lavender’ stage 3 petals, buds and leaves, the expression of RhEIL exceeded that of RhEIN3. In petals, the amount of both transcripts was maximal at stage 2 and 3 for ‘Vanilla’ but at stage 1 and 2 for ‘Lavender’. As for the parent plants, expression analysis of stage 2 petals, buds and leaves from the selected F1 plants detected both transcripts (Supplementary Fig. S4) and more RhEIL than RhEIN3 transcript. There is only little variation between expression in the 4 genotypes. For the petals, the total expressions are similar to that of stage 2 petals from the parent plants, and for leaves the total expressions are also similar. 3.4. Ethylene biosynthetic genes (RhACS1-5) Expression of the ethylene biosynthetic enzyme genes RhACS15 was analyzed in ‘Vanilla’ and ‘Lavender’. Whereas RhACS1 and RhACS2 transcripts were detected in all investigated tissues for both cultivars (Fig. 5), RhACS3-5 transcripts were not detected in any of
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Fig. 2. Expression patterns for RhETR1, RhETR2 and RhETR3 at three developmental stages of (carpels and petals), and in floral buds and leaves of ‘Vanilla’ and ‘Lavender’ as determined by RT-PCR. Each column represents the optical density of the PCR band divided by the optical density of the band for the internal standard Rhˇ-actin, with correction for difference in amplicon sizes. The values are given as means of densitometric units of three RT-PCR reactions; error bars represent standard error of the mean. The numbers at the top are the sum of the heights of the columns for RhETR1-3.
Fig. 3. Expression patterns for RhCTR1 and RhCTR2 at three developmental stages of (carpels and petals), and in floral buds and leaves of ‘Vanilla’ and ‘Lavender’ as determined by RT-PCR. Each column represents the optical density of the PCR band divided by the optical density of the band for the internal standard Rhˇ-actin, with correction for difference in amplicon sizes. The values are given as means of densitometric units of three RT-PCR reactions; error bars represent standard error of the mean. The numbers at the top are the sum of the heights of the columns for RhCTR1 and RhCTR2.
the investigated tissues (data not shown). In ‘Vanilla’ and ‘Lavender’ carpels and in ‘Vanilla’ petals, buds and leaves RhACS2 was more expressed than RhACS1, whereas the opposite was the case for ‘Lavender’ petals (except for stage 1), buds and leaves. The sum of the two transcript levels is in all cases larger for ‘Vanilla’ than for ‘Lavender’. As for the parent plants, expression analysis of stage 2 petals, buds and leaves from the selected F1 plants detected RhACS1 and RhACS2 transcripts (Supplementary Fig. S5), but no RhACS3, RhACS4 and RhACS5 transcripts (data not shown), in the investigated tissues. The expression of RhACS1 was always higher than the RhACS2 expression in petal tissue, as for ‘Lavender’ but not for ‘Vanilla’ (Fig. 5), regardless of sensitivity to ethylene (Supplementary Fig. S5). In addition, the sum of the two transcript levels in petals is larger than for both ‘Lavender’ and ‘Vanilla’ for all 4 genotypes. For leaves and buds the patterns were more variable, but without conspicuous correlation with ethylene sensitivity.
4. Discussion To clarify the reasons for the significant differences in postharvest characteristics of miniature potted roses, mRNA expression patterns of genes involved in ethylene biosynthesis, perception and signal transduction pathways were investigated and compared for the cultivars ‘Vanilla’ and ‘Lavender’, which show low and high ethylene sensitivity, respectively, and plants selected among their F1 offspring for low and high ethylene sensitivity (Section 2.1). Based on the current model of ethylene signal transduction (briefly described in the Introduction), and the experimental evidence behind it, it may be expected that plants with increased levels of receptor protein, increased CTR1 levels, decreased EIN2 levels, or decreased EIN3/EIL levels should show decreased ethylene sensitivity. We investigated the transcript levels for the cloned Rosa hybrida genes for ethylene signal transduction proteins in the above-mentioned cultivars to look for a possible correlation
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Fig. 4. Expression patterns for RhEIN3 and RhEIL at three developmental stages of (carpels and petals), and in floral buds and leaves of ‘Vanilla’ and ‘Lavender’ as determined by RT-PCR. Each column represents the optical density of the PCR band divided by the optical density of the band for the internal standard Rhˇ-actin, with correction for difference in amplicon sizes. The values are given as means of densitometric units of three RT-PCR reactions; error bars represent standard error of the mean. The numbers at the top are the sum of the heights of the columns for RhEIN3 and RhEIL.
Fig. 5. Expression patterns for RhACS1 and RhACS2 at three developmental stages of (carpels and petals), and in floral buds and leaves of ‘Vanilla’ and ‘Lavender’ as determined by RT-PCR. Each column represents the optical density of the PCR band divided by the optical density of the band for the internal standard Rhˇ-actin, with correction for difference in amplicon sizes. The values are given as means of densitometric units of three RT-PCR reactions; error bars represent standard error of the mean. The numbers at the top are the sum of the heights of the columns for RhACS1 and RhACS2.
between transcript levels and ethylene sensitivity that could confirm (or disagree with) these expectations. RhEIN2 is not yet cloned and was therefore not investigated. Müller et al. (2000a) reported that the expression of RhETR1 in petals from the cultivar ‘Bronze’ with short flower life was clearly higher than for the long-lasting ‘Vanilla’, in contrast to what could be expected from the ethylene signal transduction model. We found, however, that the amount of RhETR1 mRNA in ‘Vanilla’ was higher than for the short-lasting cultivar ‘Lavender’ at all developmental stages in carpels, at stage 3 in petals, and in buds (Fig. 2). In floral buds and in leaves the levels of all three receptor transcripts were higher for ‘Vanilla’ than for ‘Lavender’ (Fig. 2). In addition, the total amount of transcript for RhETR1-3 was higher for ‘Vanilla’ than for ‘Lavender’ in all cases except for stage 1 petals and leaves. These findings are what would be expected from the signal transduction pathway. The expression of RhETR1 in F1 progeny plants of ‘Vanilla’ and ‘Lavender’ was evaluated for petals, floral buds, and
leaves (Supplementary Fig. S2). RhETR1 was expressed in all tissues, and no clear differences were seen between the long-lasting genotypes 48 and 131 and the short-lasting genotypes 50 and 74. The long-lasting genotypes, however, had significantly lower total receptor expression in buds and leaves (but not in petals) than the short-lasting genotypes, which is not what could be expected from the model of ethylene signal transduction. For ‘Vanilla’, in contrast, the total receptor expression in buds is larger than for ‘Lavender’ buds (Fig. 2). In petals, the amount of RhETR2 mRNA was higher for ‘Vanilla’ than for ‘Lavender’ at stage 2 and 3 and all three transcripts reached their maximum at stage 3 for ‘Vanilla’ but stage 2 for ‘Lavender’ (Fig. 2). RhETR2 expression was higher than RhETR1 expression for ‘Vanilla’ petals, ‘Lavender’ stage 3 petals and ‘Lavender’ buds, whereas the opposite was the case for the other analyzed tissues (Fig. 2). These results for petal RhETR2 transcripts are not in agreement with observations by Müller et al. (2000b), who found that
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the expression of RhETR2 in petals of the two cultivars ‘Bronze’ and ‘Vanilla’ is constitutive during flower senescence, and that the transcript abundance was higher in ‘Bronze’ (more ethylene sensitive) than in ‘Vanilla’ (less ethylene sensitive) at all three stages of development. RhETR2 was expressed in all analyzed tissues of the selected F1 plants (Fig. 2). Remarkably, RhETR2 was significantly more expressed than RhETR1 or RhETR3, except for genotype 48 buds. The expression of RhETR3 was lower than 0.2 (in many cases lower than 0.1) in all analyzed ‘Vanilla’ and ‘Lavender’ tissues, except for ‘Vanilla’ leaves (Fig. 2). The same was the case for the F1 genotypes (Supplementary Fig. S2). Müller et al. (2000b) reported that the level of mRNA for RhETR3 in petals of ‘Bronze’ was clearly higher than for ‘Vanilla’. In contrast to this observation, the RhETR3 mRNA level for stage 3 ‘Vanilla’ petals is higher than the level for stage 3 ‘Lavender’ petals and it was not expressed in buds and leaves of ‘Lavender’ (Fig. 2). Since there are some differences in RhETR3 expression between ‘Vanilla’ and ‘Lavender’, further research is required to clarify the relation between the RhETR3 transcript level and ethylene sensitivity. There was no detectable expression of RhETR4 in any of the analyzed ‘Lavender’ and ‘Vanilla’ or F1 plant tissues. In accordance with these observations, Xue et al. (2008) did not detect RhETR4 expression in gynoecia, petals, stamens, sepals, and receptacles of the rose cultivar ‘Samantha’. Müller et al. (2000b) isolated a 547 bp RhETR4 sequence. The amino acid sequence deduced from this internal fragment of RhETR4 is 79% identical to that for RhETR1. However, the authors did not investigate the RhETR4 expression. Expression of the RhCTR1 and RhCTR2 genes, which encode protein kinases that inhibit EIN2 when ethylene is not bound to the receptors, were analyzed. RhCTR2 was significantly more expressed than RhCTR1 in all investigated tissues, except for ‘Lavender’ stage 3 carpels where both showed very low expression. It should also be noted that for petals there is an increase in total expression when petals develop from stage 2 to stage 3 for ‘Vanilla’ but a decrease for ‘Lavender’ (Fig. 3). Hajizadeh et al. (2011) obtained similar results as they observed that expression of RhCTR1 increased during flower opening and that expression of RhCTR1 was higher in a cultivar with long vase life than in one with short vase life. Müller et al. (2002, 2003) observed no expression differences of RhCTR1 and RhCTR2 between the long-lasting ‘Vanilla’ and the short-lasting ‘Bronze’ cultivar, but found an increase of RhCTR1 expression in petals during flower development. The higher level of total expression in ‘Vanilla’ than in ‘Lavender’ stage 3 carpels and petals is in accordance with the standard model for the ethylene signal transduction pathway, since that predicts that higher CTR expression should result in less ethylene sensitive plants. The RhEIN3 and RhEIL genes encode transcription factors that, in the presence of ethylene, activates the transcriptional response to ethylene. RhEIN3 and RhEIL expression was detected in all investigated tissues (Figs. 4 and S4). In all cases, except for a few tissues from the parent plants, the expression of RhEIL exceeded that of RhEIN3. For the F1 plants, there was only little variation between expression in the 4 genotypes, and the total expressions in petals were similar to that of stage 2 petals from the parent plants. For the 3 stages of ‘Vanilla’ and ‘Lavender’ petals the expression levels are relatively constant. In accordance with these observations, Müller et al. (2003) reported constitutive and stable expression of RhEIN3 in petals of ‘Vanilla’ and ‘Bronze’ cultivars during flower senescence, and Ma et al. (2006) reported constant expression of Rh-EIN3-1 (here termed RhEIN3) and Rh-EIN3-2 (here termed RhEIL) in cut ‘Samantha’ roses. Consequently, if there are differences in RhEIN3 and RhEIL activity in cultivars showing different ethylene sensitivity, they are probably mostly due to posttranscriptional control such as control of protein stability (Cho and Yoo 2015).
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High susceptibility to ethylene may possibly be due to induction of increased activity of ethylene biosynthetic enzymes by exposure to external ethylene. In higher plants, ethylene biosynthesis has been well characterized (Yang and Hoffman, 1984), and many studies have been performed on regulation of the enzymes involved (Xu and Zhang 2015). ACC synthase (ACS) and ACC oxidase (ACO), the two key enzymes in the ethylene biosynthetic pathway, are regulated both transcriptionally and post transcriptionally (Xu and Zhang 2015). Thain et al. (2004) showed, eg., that expression of ACC synthase genes in Arabidopsis is controlled by light, by the circadian clock, and by negative feedback regulation through ethylene signaling. Since ACS is generally considered the rate-limiting enzyme of ethylene biosynthesis we decided to study the 5 ACC synthase genes for which rose sequences were available. The RT-PCR result showed that only RhACS1 and RhACS2 were expressed in the investigated tissues of ‘Vanilla’, ‘Lavender’ and the selected F1 plants, while RhACS3, RhACS4 and RhACS5 expression could not be detected. The sum of the two transcript levels is in all cases larger for ‘Vanilla’ than for ‘Lavender’ (Fig. 5). This is contrary to what could be expected if it is assumed that transcript level correlates with enzyme activity and that ethylene production in the tissues contributes to a lower ethylene sensitivity. For the F1 plants, expression of RhACS1 was always higher than the RhACS2 expression in petal tissue (Supplementary Fig. S5). In addition, the sum of the two transcript levels in petals is larger than for both ‘Lavender’ and ‘Vanilla’ for all 4 genotypes. Again, there is no obvious correlation between transcript levels and ethylene sensitivity. Müller et al. (2000a) found that the ACC synthase transcript (detected by northern-blotting and use of a Pelargonium ACC synthase probe) increased during flower senescence in ‘Vanilla’ but remained constant at a low level in ‘Bronze’. That the level was higher in ‘Vanilla’ than in ‘Bronze’ agrees with our finding of a higher total expression in ‘Vanilla’ than in ‘Lavender’ petals (Fig. 5). Liu et al. (2013) reported RhACS1-3 expression in cut ‘Samantha’ roses throughout the dehydration period. They also reported that RhACS4 and RhACS5 expression could hardly be detected. A recent study of Khan et al. (2015) showed that the promoter activities of RhACS1 and RhACS2 in ‘Samantha’ were strong during the development from young seedlings to mature flowering plants in various investigated organs, including hypocotyls, cotyledons, leaves, roots and lateral roots. Except for the RhACS3 expression, these results agree with those obtained for ‘Vanilla’, ‘Lavender’ and their F1 progeny. From the observations in the present work it is not possible to find a clear correlation between transcript levels of the analyzed genes and the ethylene-sensitivity of the studied rose cultivars. A lack of clear correlation between transcription of ethylene signal transduction genes and ethylene sensitivity was also noted by Ahmadi et al. (2009), but it was established for more genes in this study. These observations indicate that much of the variation seen for transcript levels may be due to random factors (such as variation in the genotypes and epigenetic state of the analyzed plants) that are not important for ethylene sensitivity, and that other genes or factors could more tightly regulate the sensitivity to ethylene. In accordance with this, recent molecular investigations of the ethylene signal transduction pathway have revealed other processes, such as proteasomal degradation and MAP kinase cascades, that are involved in the signal processes (Cho and Yoo 2015). It is therefore likely that transcription of other genes involved in ethylene signal transduction, posttranscriptional or posttranslational control, or crosstalk with other signal transduction pathways may influence the degree of ethylene sensitivity of miniature roses.
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Acknowledgements The experimental work was partly supported by a PhD grant from the German Academic Exchange Service (DAAD) (Ref.: 412, PKZ: A/03/33925). The authors would like to thank Prof. em. Bjarne M. Stummann (University of Copenhagen, Denmark) for his valuable advices and critical review of the manuscript. Authors would also like to thank technical and scientific staff: Dr. Heiko Mibus, Mrs. Annette Steding and Mr. Herbert Geyer, from the Section of Floriculture at Leibniz University of Hannover for fruitful discussions and technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2016. 12.029. References Ahmadi, N., Mibus, H., Serek, M., 2009. Characterization of ethylene-induced organ abscission in F1 breeding lines of miniature roses (Rosa hybrida L.). Postharvest Biol. Technol. 52, 260–266. Andersen, L., Williams, M.H., Serek, M., 2004. Reduced water availability improves drought tolerance of potted miniature roses; Is the ethylene pathway involved? J. Hortic. Sci. Biotech. 79, 1–13. Buanong, M., Mibus, H., Sisler, E.C., Serek, M., 2005. Efficacy of new inhibitors of ethylene perception in improvement of display quality of miniature potted roses (Rosa hybrida L.). Plant Growth Regul. 47, 29–38. Cancel, J.D., Larsen, P.B., 2002. Loss-of-Function mutations in the ethylene ReceptorETR1 cause enhanced sensitivity and exaggerated response to ethylene in arabidopsis. Plant Physiol. 129, 1557–1567. Cho, Y.H., Yoo, S.D., 2015. Novel connections and gaps in ethylene signaling from the ER membrane to the nucleus. Front. Plant Sci. 5, 1–7. Ferrante, A., Trivellini, A., Scuderi, D., Romano, D., Vernieri, P., 2015. Post-production physiology and handling of ornamental potted plants. Postharvest Biol. Technol. 100, 99–108. FloraHolland, 2016. Flora Holland: Our Facts & Figures Annual Report 2015. Koninklijke Coöperatieve Bloemenveiling Flora Holland U.A.16. Gallie, D.R., 2015. Ethylene receptors in plants-why so much complexity? F1000Prime Reports 7, 1–12. Hajizadeh, H., Razavi, K., Mostofi, Y., Cacco, G., Mousavi, A., Zamani, Z., Stevanato, P., 2011. Expression of genes encoding protein kinases during flower opening in two cut rose cultivars with different longevity. Iran. J. Biotechnol. 9, 230–233. Ju, C., Yoon, G.M., Shemansky, J.M., Lin, D.Y., Ying, Z.I., Chang, J., Garrett, W.M., Kessenbrock, M., Groth, G., Tucker, M.L., Cooper, B., 2012. CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proc. Natl Acad. Sci 109, 19486–19491. Khan, M.A., Meng, Y., Liu, D., Tang, H., Lü, S., Imtiaz, M., Jiang, G., Lü, P., Ji, Y., Gao, J., Ma, N., 2015. Responses of rose RhACS1 and RhACS2 promoters to abiotic stresses in transgenic Arabidopsis thaliana. Plant Cell Rep. 34, 795–804. Liu, D., Liu, X., Meng, Y., Sun, C., Tang, H., Jiang, Y., Khan, M.A., Xue, J., Ma, N., Gao, J., 2013. An organ-specific role for ethylene in rose petal expansion during dehydration and rehydration. J. Exp. Bot. 64, 2333–2344. Müller, R., Andersen, A.S., Serek, M., 1998. Differences in display life of miniature potted roses (Rosa hybrida L.). Sci. Hortic. 76, 59–71. Müller, R., Lind-Iversen, S., Stummann, B.M., Serek, M., 2000a. Expression of genes for ethylene biosynthetic enzymes and an ethylene receptor in senescing flowers of miniature roses. J. Hortic. Sci. Biotech. 75, 12–18.
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